Zinc fingers belonging to the Cys2-His2 family constitute one of the most common DNA-binding motifs found in eukaryotes, and these zinc fingers have provided a very attractive framework for the design and selection of DNA-binding proteins with novel sequence specificities. Numerous studies have used phage display methods or design ideas to explore and systematically alter the specificity of zinc finger-DNA interactions (Desjarlais and Berg, Proteins Struct. Funct. Genet. 12:101-104 (1992); Desjarlais and Berg, Proc. Natl. Acad. Sci. USA 90:2256-2260 (1993); Rebar and Pabo, Science 263:671-673 (1994); Jamieson et al., Biochemistry 33:5689-5695 (1994); Choo and Klug, Proc. Natl. Acad. Sci. USA 91:11163-11167 (1994); Wu et al., Proc. Natl. Acad. Sci. USA 92:344-348 (1995); and Greisman and Pabo, Science 275:657-661 (1997)).
Structure based computer design has been used to link Cys2-His2 zinc fingers with other DNA-binding domains, including other zinc finger proteins, to generate hybrid proteins that recognize extended sites (Pomerantz et al., Science 267:93-96 (1995); Kim et al, Proc. Natl. Acad. Sci. USA 94:3616-3620 (1997)). For example, zinc finger proteins have been linked to a GAL4 dimerization domain to develop novel homo- and hetero-dimers (Pomerantz et al., Biochemistry 4:965-970 (1997)), and to a nuclease domain to generate novel restriction enzymes (Kim et al., Proc. Natl. Acad. Sci. USA 93:1156-1160 (1996)). Zinc finger/homeodomain fusion is being tested for potential applications in gene therapy (Rivera et al., Nature Med. 2:1028-1032 (1996)).
There also have been several attempts to increase affinity and specificity of zinc finger proteins by adding additional fingers to a three-finger protein (Rebar, (Ph.D. Thesis), Selection Studies of Zinc Finger-NA Recognition, Massachusetts Institute of Technology (1997); Shi, Y. (Ph.D. Thesis) Molecular Mechanisms of Zinc Finger Protein-Nucleic Acid Interactions, Johns Hopkins University (1995)) or by tandemly linking two three-finger proteins (Liu et al., Proc. Natl. Acad. Sci. USA 94:5525-5530 (1997)). However, these previous design strategies for poly-finger proteins, which all used canonical xe2x80x9cTGEKPxe2x80x9d linkers (linkers having the amino acid sequence threonine-glycine-glutamate-lysine-proline; SEQ ID NO:13) to connect the additional fingers, resulted in relatively modest increases in affinity. There is thus a need to develop linkers that provide enhanced affinity and specificity to chimeric zinc finger proteins.
The present invention therefore provides a method of using structure based design to select flexible linkers and make chimeric zinc finger proteins with enhanced affinity and specificity. The present invention also provides a method of making chimeric zinc finger proteins that have flexible linkers of 5 amino acids or more in length to make chimeric zinc finger proteins with enhanced affinity and specificity. Zinc finger proteins made using these methods have binding affinities in the femtomolar range and provide, e.g., high levels (more than about 70 fold) of transcriptional repression at a single target site. Such zinc finger proteins can be used for regulation of gene expression, e.g., as therapeutics, diagnostics, and for research applications such as functional genomics.
In one aspect, the present invention provides a method of making a chimeric zinc finger protein that binds to adjacent target sites, the method comprising the steps of: (i) selecting a first and a second DNA-binding domain polypeptide of the chimeric zinc finger protein, wherein at least one of the domains comprises a zinc finger polypeptide, and wherein the first domain binds to a first target site and the second domain binds to a second target site, which target sites are adjacent; (ii) using structure-based design to determine the physical separation between the first and second domains when they are individually bound to the first and second target sites; (iii) selecting a flexible linker that is at least 1-2 xc3x85 longer than the physical separation between the first and second domains; and (iv) fusing the first and second domains with the flexible linker, thereby making a chimeric zinc finger protein that binds to adjacent target sites.
In another aspect, the present invention provides a method of making a chimeric zinc finger protein that binds to adjacent target sites, the method comprising the steps of: (i) selecting a first and a second DNA-binding domain polypeptide of the chimeric zinc finger protein, wherein at least one of the domains comprises a zinc finger polypeptide, and wherein the first domain binds to a first target site and the second domain binds to a second target site, which target sites are adjacent; (ii) selecting a flexible linker that is five or more amino acids in length; and (iv) fusing the first and second domains with the flexible linker, thereby making a chimeric zinc finger protein that binds to adjacent target sites.
In another aspect, the present invention provides a chimeric zinc finger protein that binds to adjacent target sites, the chimeric zinc finger protein comprising: (i) a first and a second DNA-binding domain polypeptide of the chimeric zinc finger protein, wherein at least one of the domains comprises a zinc finger polypeptide, and wherein the first domain binds to a first target site and the second domain binds to a second target site, which target sites are adjacent; and (ii) a flexible linker that is at least 1-2 xc3x85 longer than the physical separation between the first and second domains when they are individually bound to the first and second target sites, as determined by structure-based modeling; wherein the first and second domains are fused with the flexible linker.
In another aspect, the present invention provides a chimeric zinc finger protein that binds to adjacent target sites, the chimeric zinc finger protein comprising: (i) a first and a second DNA-binding domain polypeptide of the chimeric zinc finger protein, wherein at least one of the domains comprises a zinc finger polypeptide, and wherein the first domain binds to a first target site and the second domain binds to a second target site, which target sites are adjacent; and (ii) a flexible linker that is five or more amino acids in length; wherein the first and second domains are fused with the flexible linker.
In one embodiment, the present invention provides nucleic acids encoding the chimeric zinc finger proteins.
In one embodiment, the first and the second domains are zinc finger polypeptides. In another embodiment, the zinc finger polypeptide is selected from the group consisting of Zif268 and NRE. In another embodiment, the zinc finger polypeptides are heterologous. In one embodiment, the first domain is a zinc finger polypeptide and the second domain comprises a heterologous DNA-binding domain polypeptide. In another embodiment, the chimeric zinc finger protein further comprises a regulatory domain polypeptide.
In one embodiment, the chimeric zinc finger protein has femtomolar affinity for the adjacent target sites. In another embodiment, the chimeric zinc finger protein has about 2-4 femtomolar affinity for the adjacent target sites.
In one embodiment, the flexible linker is 5, 8, or 11 amino acids in length. In another embodiment, the flexible linker has the sequence RQKDGERP (SEQ ID NO:14) or RQKDGGGSERP (SEQ ID NO:15).
In one embodiment, the target sites are separated by one or two nucleotides.
In one embodiment, the adjacent target sites are separated by zero nucleotides and the flexible linker is five or six amino acids in length. In another embodiment, the adjacent target sites are separated by one nucleotide and the flexible linker is seven, eight, or nine amino acids in length. In another embodiment, the adjacent target sites are separated by two nucleotides and the flexible linker is ten, eleven, or twelve amino acids in length. In another embodiment, the adjacent target sites are separated by three nucleotides and the flexible linker is twelve or more amino acids in length.